Helicobacter pylori (also designated as H.pylori) is a Gram-negative bacterium which, to date, has been found exclusively on the surface of the mucosa of the stomach in man.
In common with most bacteria, H.pylori is sensitive to a medium which is at acid pH but, nevertheless, is able to tolerate acidity in the presence of physiological concentrations of urea (Marshall et al. (1990) Gastroenterol. 99: 697-702). By hydrolysing the urea to form carbon dioxide and ammonia, which are released into the microenvironment of the bacterium, the H.pylori urease enables the bacterium to survive in the acidic environment of the stomach. Recently, studies carried out on animal models have provided data suggesting that the urease is an important factor in the colonization of the gastric mucosa (Eaton et al. (1991) Infect. Immun. 59: 2470-2475). The urease is also suspected of causing injury, either directly or indirectly, to the gastric mucosa.
Currently, Helicobacter pylori (H.pylori) is recognized as being the etiological agent of antral gastrites, and appears to be one of the cofactors required for the development of ulcers. Furthermore, it appears that the development of gastric carcinomas may be associated with the presence of H.pylori. 
In order to develop novel sensitive and specific means for detecting in-vitro infections due to bacteria of the Helicobacter pylori species, the inventors have been taking an interest in the system for regulating the mobility of these bacteria.
With this aim in view, they have been interested in different modifications of the H.pylori strains, modifications which did not affect the recognition of these bacteria by sera from infected patients but which nevertheless rendered it possible to avoid obtaining reactions of the xe2x80x9cfalse positivexe2x80x9d type, in particular with bacteria of the Campylobacter family, for example Campylobacter jejuni. 
Furthermore, the inventors observed that it was possible, if need be, for the modified bacteria which were obtained to be employed in constructing immunogenic compositions or compositions used for vaccination. In this respect, the invention proposes, in particular, live attenuated bacterial strains.
In a first step, the inventors identified and isolated the gene f1bA which is involved in the regulation of the biosynthesis of the flagella of H.pylori and, as a consequence, in the regulation of the mobility of the bacterium. The biosynthesis of the flagella comprises synthesizing flagellins A and B and synthesizing the sheath. The f1bA gene regulates both the synthesis of flagellins A and B and the synthesis of the sheath which contains these flagellins. The inventors established that the f1bA gene was also important in that it regulated the biosynthesis of the anchoring protein of the bacterium, also termed the xe2x80x9chookxe2x80x9d.
The invention therefore relates to a nucleotide sequence from the f1bA gene regulating the biosynthesis of the proteins of the Helicobacter pylori flagella, characterized in that it is able to hybridize, under conditions of high stringency, with a probe corresponding to a nucleotide fragment from H. pylori which has been amplified using two oligonucleotides having the following sequences:
OLF1bA-1: ATGCCTCGAGGTCGAAAAGCAAGATG (SEQ ID NO:1).
OLF1bA-2: GAAATCTTCATACTGGCAGCTCCAGTC (SEQ ID NO:2), or able to hybridize, under conditions of high stringency, with these oligonucleotides.
Such a sequence can be obtained by the steps of:
screening a genomic library containing the chromosomal DNA of an H. pylori strain with a probe corresponding to a nucleotide fragment from H. pylori which has been amplified using two oligonucleotides having the following sequences:
OLF1bA-1: ATGCCTCGAGGTCGAAAAGCAAGATG (SEQ ID NO:1).
OLF1bA-2: GAAATCTTCATACTGGCAGCTCCAGTC (SEQ ID NO:2), or able to hybridize, under conditions of high stringency, with these oligonucleotides,
recovering the DNA sequences which hybridize with said probe,
subcloning the DNA sequences which have been obtained in an appropriate vector of the plasmid type and selecting those modified vectors which hybridize, under conditions of high stringency, with the probe corresponding to the DNA fragment from H. pylori which has been amplified using oligonucleotides OLF1bA-1 and OLF1bA-2,
sequencing the DNA fragments contained in the plasmid vectors which hybridize with the abovementioned probe and determining the open reading frame contained in these fragments.
Advantageously, these DNA fragments will be used to reconstitute the coding sequence of the f1bA gene, corresponding to an open reading frame comprising approximately 2196 nucleotides.
The genomic library containing the chromosomal DNA of H.pylori can be obtained from any H.pylori strain. A cosmid library may also be prepared from the chromosomal DNA of H.pylori. 
An example of a strain which can be used for constructing this library is the strain N6, which was deposited in the NCIMB on Jun. 26, 1992 under No. NCIMB40512.
The two oligonucleotide primers which are used for preparing the probe which is intended for hybridizing the sought-after DNA which is present in the H.pylori DNA library are selected from the conserved regions of the various proteins of the LcrD/F1bF family.
The two oligonucleotide primers, OLF1bA-1 and OLF1bA-2, enabled a fragment to be amplified which was usable as a probe and which was of 130 base pairs, having the following sequence:
The conditions of high stringency referred to above are the following: the hybridization is carried out at 42xc2x0 C. in the presence of 50% formamide in a 2xc3x97SSC buffer containing 0.1% SDS (1xc3x97SSC corresponds to 0.15 M NaCl plus 15 mM sodium citratexe2x80x94pH 7.0). The washings are carried out at 68xc2x0 C., for example twice during a period of one hour, using 2xc3x97SSC plus 0.1% SDS.
A nucleotide sequence which is particularly interesting in accordance with the invention is the sequence of the f1bA gene corresponding to the sequence of nucleotides depicted in FIG. 2 (SEQ ID NO:6), or to a nucleotide sequence which hybridizes, under conditions of high stringency, with the abovementioned sequence.
According to another embodiment of the invention, the nucleotide sequence which is the subject-matter of the present application is characterized in that it encodes a protein having the amino acid sequence (SEQ ID NO:7) depicted in FIG. 2 or an amino acid sequence possessing the same regulatory properties, with regard to the biosynthesis of the flagellar proteins of H.pylori, as the abovementioned sequence.
The invention also relates to a nucleotide sequence which corresponds to the previous definitions and which is modified by deletion, substitution or insertion of bases or of a fragment of a nucleotide sequence, such that:
either the f1bA gene is no longer expressed in a host cell,
or the expression of the f1bA gene in a host cell does not enable the A and B flagellins or the sheath which contains them to be biosynthesized and, if this is the case, does not enable the H.pylori anchoring protein or the hook, to be synthesized.
The modification to which the nucleotide sequence of the invention is subjected should be such that it is irreversible and, in particular, that it remains irreversible when this sequence is recombined with the f1bA gene which is present in a bacterium which is transformed with a nucleotide sequence which is modified in this manner. This recombination is, for example, of the xe2x80x9cdouble crossing overxe2x80x9d type. Preferably, the modification of the nucleotide sequence should not involve any substantial modificationxe2x80x94after replacement, by this modified sequence, of the corresponding fragment of the normal f1bA gene in a given H.pylori strainxe2x80x94of the functions of the neighbouring genes.
Also included within the scope of the invention are nucleotide sequences which constitute a fragment of the f1bA gene meeting the above criteria. As examples, fragments which are the subject-matter of the invention consist of at least 6 nucleotide sequences, preferably at least 50, if not at least 100 nucleotides.
Such fragments are, for example, selected either on account of their specific f1bA gene character or because they belong to conserved regions of several genes encoding proteins of the LcrD/F1bF family.
According to another embodiment, the invention is also directed towards the fragments of the f1bA gene which are delimited by the restriction sites which are present in the gene. Some of these sites are defined, by way of example, in FIG. 1B.
Another fragment according to the invention is a fragment of at least 1000 bp which is derived from any region of the f1bA gene and which preferably includes a restriction site or is capable of accommodating a restriction site.
Other nucleotide sequences of the invention are, for example, recombinant nucleic acids which comprise a nucleotide sequence such as those which have been described above, itself modified by the insertion of a cassette containing a marker, for example a gene for resistance to an antibiotic or a gene for resistance to a heavy metal such as described in Application FR 9406202, which was filed on May 20, 1994.
Thus, a cassette for resistance to kanamycin can be inserted. Various techniques can be used in this context and reference is made, in particular, to the paper of Labigne A. et al. (J. of Bacteriology, Vol. 170, 1988, p. 1704-1708) and the paper of Labigne A. et al. (Res. Microbiol 1992, 143, 15-26).
The invention also relates to specific oligonucleotides from a previously defined nucleotide sequence, which oligonucleotides are characterized in that they possess one of the following sequences:
OLF1bA-1: ATGCCTCGAGGTCGAAAAGCAAGATG (SEQ ID NO:1).
OLF1bA-2: GAAATCTTCATACTGGCAGCTCCAGTC (SEQ ID NO:2).
OLF1bA-7: CGGGATCCGTGGTTACTAATGGTTCTAC (SEQ ID NO:4).
OLF1bA-8: CGGGATCCTCATGGCCTCTTCAGAGACC (SEQ ID NO:5).
According to another embodiment, the invention relates to an amino acid sequence from the F1bA protein of H.pylori, which sequence is characterized in that it is encoded by a nucleotide sequence such as previously defined.
A specific amino acid sequence (SEQ ID NO:7) from the F1bA protein of H.pylori is depicted in FIG. 2.
Thus, within the scope of the invention, the f1bA gene and the protein expressed by this gene can be of interest, in particular for employment in immunogenic compositions or compositions used for vaccination.
The invention is also directed towards bacterial strains of Helicobacter pylori which possess an aflagellate phenotype, which phenotype results from the mutation, by substitution, addition and/or deletion of bases or of a nucleotide fragment, of the above-defined nucleotide sequence of the f1bA gene involved in the regulation of the biosynthesis of the flagellar proteins of H.pylori. 
This modification of the f1bA gene makes it possible to obtain a strain of the aflagellate type, that is which no longer expresses the F1aA and F1aB proteins and which preferably no longer expresses the proteins of the sheath.
According to one embodiment of this bacterial strain, the strain which is obtained additionally lacks the hook protein of H.pylori. 
Preferably, a bacterial strain which meets the abovementioned criteria is characterized in that it is obtained from the strain N6, which was deposited in the NCIMB on Jun. 26, 1992 under number NCIMB 40512.
By way of example, the invention relates to a recombinant aflagellate strain of H.pylori which is designated N6f1bAxe2x88x92and was deposited in the NCIMB on Jun. 30, 1995 under the No. NCIMB 40747.
Such aflagellate strains of H.pylori are of particular interest for employment in serology and, as a consequence, for the in-vitro detection of an infection due to H.pylori. These strains are advantageously of the recombinant type.
In particular, these strains exhibit the advantage of enabling an infection due to H.pylori to be detected in vitro in a specific and sensitive manner. In other words, the invention advantageously enables an infection due to H.pylori to be detected specifically while avoiding, in particular, xe2x80x9cfalse-positivexe2x80x9d results, for example with bacterial strains such as Salmonella or Campylobacter.
Given that the strains of H.pylori of the aflagellate type, which have thus been defined, may also have other applications, for example may be employed in the preparation of vaccine compositions, there can be interest in preparing recombinant aflagellate bacterial strains which possess a second modification or mutation, for example an aflagellate bacterial strain can be prepared which is characterized in that it is additionally mutated in such a way that it produces an attenuated urease, or even no longer produces urease, with the mutation consisting, for example, of a mutation of the nucleotide sequence of one or more genes selected from among the genes ureA, ureB, ureC, ureD, ureE, ureF, ureG, ureH or ureI. The urease structural genes, designated ureA, ureB, ureC and ureD of urease, have been described in the publication (Labigne et al (1991) J. Bacteriol. 173: 1920-1931). The other genes have been described in Patent Application EP 0610322.
The bacterial strains of the invention may be employed as such or in extract form, and, in particular, the invention relates to a total bacterial strain extract which is obtained from the previously described strains.
Such a bacterial extract can be prepared by extracting with n-octyl glucoside. In this case, the preparation technique which is employed is that described by LELWALA-GURUGE J. (Scand. J. Infect. Dis. 1992, 24: 457-465).
Another bacterial extract can be obtained by extracting with PBS or glycine using the techniques described, respectively, by BAZILLOU M. et al (Clin. Diagn. Lab. Immuno., 1994, 1: 310-317) and AGUIRRE P.M. (Eur. J. Clin. Microbiol. Infect. Dis., 1992, 11: 634-639).
Within the scope of these applications, the invention relates to a composition for the in-vitro detection of an infection due to H.pylori in a sample of biological fluid obtained from a patient, in particular in a sample of serum, which composition includes, as the active principle, a bacterial strain of the invention or a bacterial extract in accordance with the description given above.
The biological samples which are used may be of any type and can, in particular, be any type of biological fluid, such as serum, saliva or urine, for example.
In the same way, the techniques which are employed for the detection are any techniques which involve reactions of the immunological type, in particular of the antigen/antibody type. For example, use is made of techniques such as Western blot, ELISA, etc.
The invention also relates, therefore, to a method for the in-vitro detection of an infection due to H.pylori in a sample of biological fluid taken from a patient, in particular in a sample of serum, which method comprises the steps of:
bringing the sample under test into contact with a bacterial strain according to the invention or with a bacterial extract as defined above,
detecting an immunological reaction between the said bacterial strain and antibodies which are directed against H.pylori and which are present in the sample under test.
By way of example, an in-vitro detection on a biological sample in order to look for an infection due to H.pylori can be carried out by implementing the following steps:
plates are covered with the antigen which is used for the detection and which may be a pure or recombinant protein or else an aflagellate strain or a bacterial extract, in particular an NOG (n-octyl glucoside) extract of the N6f1bA- strain (by way of example, the quantity of extract might be 3 xcexcg/ml or the quantity of antigen might be 2 xcexcg/ml),
a range of negative and positive controls (the positive control being employed at differing dilutions) is used, and patient sera, which are diluted to 1/100, are tested in parallel (volume deposited, 100 xcexcl),
an incubation step is then carried out, for example at 37xc2x0 C. for one hour, which step is followed by several successive washings and by a further incubation, for example at 37xc2x0 C. for 1 hour, with a monoclonal conjugate (of the human IgG type labelled with peroxidase), which conjugate is employed at differing dilutions (for example at a dilution of 1/32000 in the case of an antigen and at a dilution of 1/64000 in the case of a bacterial extract), with the deposited volume being 100 xcexcl,
after the incubation with the monoclonal conjugate, several different washings are carried out (for Example 4) and the enzymic reaction is developed, in the dark and for 30 minutes, using xe2x80x9cOPD+substratexe2x80x9d. The enzymic reaction is then stopped by adding H2SO4, after which the optical densities, OD""s, are read at 492 nm/620 nm.
The invention is furthermore directed to an immunogenic composition for obtaining antibodies against H.pylori, which composition is characterized in that it includes, as the active principle, a bacterial strain according to the invention or an extract of this bacterial strain.
According to one particular embodiment of the invention, an immunogenic composition for obtaining antibodies against H.pylori is characterized in that it includes an amino acid sequence from the F1bA protein.
Also included within the scope of the present invention is a vaccinating composition for obtaining antibodies which protect against an infection due to H.pylori, characterized in that it includes, as the active principle, a bacterial strain according to the invention or a bacterial extract according to the above definitions.
Another vaccinating composition for obtaining antibodies against an infection due to H.pylori is characterized in that it includes, as the active principle, antigens of the urease type, in particular antigens encoded by the genes ureA, ureB, ureC, or ureD and a protein having an amino acid sequence as defined above.
The invention also relates to monoclonal antibodies or polyclonal sera which are directed against a previously described amino acid sequence. These antibodies are obtained by techniques which are known per se, in particular by immunizing an animal with the chosen antigen, followed either by producing and recovering the antibodies which are produced and selecting those among them which specifically recognize H.pylori, or by preparing hybridomas, by fusing spleen cells from the previously immunized animal with myeloma cells, with these hybridomas then being cultured in order to obtain monoclonal antibodies, which are selected on the basis of the specificity with which they recognize the chosen H.pylori antigen.
Other monoclonal antibodies or polyclonal sera according to the invention are directed against an aflagellate H.pylori strain such as described in the preceding pages.
The invention furthermore relates to a composition for the in vitro detection of an infection due to H.pylori in a biological sample, which composition includes, as the active principle, monoclonal antibodies or a polyclonal serum which have been obtained against an H.pylori strain of the aflagellate phenotype according to the invention.
The invention also relates to nucleotide sequences, as the active principle of a medicament, which encode amino acid sequences according to the invention, which amino acid sequences are able to induce an immunogenic response in an animal or in a patient. A technique for employing nucleotide sequences as medicaments has been described by DONNELY et al 1995, Nature Medic. 1(6), pp. 583-587.